Protein interactions between the oral bacteria Fusobacterium nucleatum and Porphyromonas gingivalis in biofilm and planktonic culture

The opportunistic pathogens Fusobacterium nucleatum and Porphyromonas gingivalis are Gram-negative bacteria associated with oral biofilm and periodontal disease. Although liquid cultures are often the preferred cultivation method in microbiology, bacterial cells in biofilm adopt a profoundly different phenotype reflecting the close cell-to-cell contact compare to their planktonic counterparts. To investigate F. nucleatum and P. gingivalis interactions relevant in biofilm formation, we applied liquid chromatography-tandem mass spectrometry to determine the expressed proteome of F. nucleatum and P. gingivalis cells that were grown either as biofilm or in planktonic culture, and individually or together. The proteomic analyses detected 1,322 F. nucleatum and 966 P. gingivalis proteins. We statistically compared the proteins label-free quantitative (LFQ) intensities between biofilm and planktonic culture and identified significant changes (p-value ≤0.05) in 0,4% F. nucleatum proteins, 7% P. gingivalis proteins, and more than 14% of all proteins in the dual-species model. For both species, proteins involved in vitamin B2 (riboflavin) metabolic process had significantly increased levels in the biofilm condition. In both mono- and dual-species biofilm models, P. gingivalis increased the production of proteins functional in translation, oxidation-reduction, and amino acid metabolism, when compared to planktonic cultures. However, when we compared LFQ intensities between mono- and dual-species models, over 90% of the significantly changed P. gingivalis proteins had their levels reduced in biofilm and planktonic settings of the dual-species model. Our findings suggest that the two bacteria interact with each other at the protein level and indicate that P. gingivalis reduces the production of multiple proteins because of more favourable growth conditions provided by F. nucleatum presence. The results highlight the complex interactions of bacteria contributing to oral biofilm, which need to be considered in the design of future prevention strategies.